25-1529-42

antibody from Invitrogen Antibodies

Targeting: CTLA4 CD, CD152, CELIAC3, CTLA-4, GSE, IDDM12

Provider product page for 25-1529-42

Flow cytometry

Other assay

Antibody data

Antibody Data
Antigen structure
References [8]
Comments [0]
Validations
Flow cytometry [1]
Other assay [8]

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Product number: 25-1529-42 - Provider product page
Provider: Invitrogen Antibodies
Product name: CD152 (CTLA-4) Monoclonal Antibody (14D3), PE-Cyanine7, eBioscience™
Antibody type: Monoclonal
Antigen: Other
Description: Description: The 14D3 monoclonal antibody reacts with human CD152, also known as cytotoxic T lymphocyte antigen-4 (CTLA-4). CTLA-4, a protein with structural similarities to CD28, is expressed on activated T cells (activated B cells may also express CTLA-4) and binds the B7 family members, CD80 (B7-1) and CD86 (B7-2), with higher affinity than CD28 does. CTLA-4 and CD28 appear to deliver opposing signals to T cells: while CD28 is a potent costimulator, CTLA-4 restricts the progression of T cells to an activated state by inhibiting IL-2 secretion and cellular proliferation. The cytoplasmic portion of CTLA-4 contains ER retention motifs, resulting in intracellular localization of a large proportion of newly synthesized CTLA-4 in response to TCR signaling. The 14D3 antibody also recognizes rhesus monkey and has inhibitor activity. Applications Reported: This 14D3 antibody has been reported for use in intracellular staining followed by flow cytometric analysis. Applications Tested: This 14D3 antibody has been tested by intracellular staining and flow cytometric analysis of stimulated human peripheral blood cells using the Intracellular Fixation & Permeabilization Buffer Set (Product # 88-8824-00) and protocol. Please refer to Best Protocols: Protocol A: Two step protocol for (cytoplasmic) intracellular proteins located under the Resources Tab online. Furthermore, due to the intracellular localization of a large portion of CTLA-4, for complete detection it may be necessary to assess intracellular expression, in addition to surface expression of CTLA-4. Light sensitivity: This tandem dye is sensitive to photo-induced oxidation. Please protect this vial and stained samples from light. Fixation: Samples can be stored in IC Fixation Buffer (Product # 00-8222) (100 µL of cell sample + 100 µL of IC Fixation Buffer) or 1-step Fix/Lyse Solution (Product # 00-5333) for up to 3 days in the dark at 4°C with minimal impact on brightness and FRET efficiency/compensation. Some generalizations regarding fluorophore performance after fixation can be made, but clone specific performance should be determined empirically. Excitation: 488-561 nm; Emission: 775 nm; Laser: Blue Laser, Green Laser, Yellow-Green Laser. Filtration: 0.2 µm post-manufacturing filtered.
Reactivity: Human
Host: Mouse
Isotype: IgG
Antibody clone number: 14D3
Vial size: 100 Tests
Concentration: 5 µL/Test
Storage: 4° C, store in dark, DO NOT FREEZE!

CTLA4 protein structure - 25-1529-42 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 6 T FR exhibit an enhanced regulatory phenotype in ex vivo HIV infection. Tonsil cells were mock-, X4-, or R5-spinoculated and cultured under experimental conditions as indicated. T FR were then analysed for expression of regulatory receptors and cytokine production by intracellular cytokine staining. ( a ) Percentage of total (surface and intracellular) T FR CTLA-4 expression ( n =15). ( b ) Percentage of surface T FR LAG-3 expression ( n =8). ( c ) Production of IL-10 by T FR ( n =7). ( d ) Production of TGF-beta-1 (measured as LAP) by T FR ( n =5). The horizontal bars of each graph indicate the median value and are listed where appropriate for clarity. Statistical analyses were performed by Friedman ( a , b ) or Mann-Whitney tests ( c , d ) and significance is denoted by asterisks where * P

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Resident memory T (T RM ) and non-T RM phenotypic profiles and susceptibility of cancer stem cell (CSC) to cytotoxic T lymphocyte (CTL)-mediated killing. (A) Flow cytometry analyses of CD8, LFA-1, CD103, CD49a, CD69, PD-1, CTLA-4, TGFBR2 and VEGFR2 on CD8 + CD103 + T RM (Heu171) and CD8 + CD103 - non-T RM (H32-22) clones. Mean immunofluorescence intensity (MFI) are in parentheses. (B) Cytotoxic activity of the T RM clone (Heu171) towards autologous IGR-Heu, Heu-CSC, CSC-1 and CSC-2 target cells. Percent of specific lysis are shown at indicated effector to target (E:T) ratios. (C) Quantification of transforming growth factor (TGF)-beta in conditioned media from IGR-Heu, Heu-CSC, CSC-1 and CSC-2 by multi-analyte flow assay. Ratios of active/total TGF-beta normalized to IGR-Heu are included. Results are presented as mean+-SEM of duplicates. (D) Cytotoxicity of the non-T RM clone (H32-22) towards autologous IGR-Heu, Heu-CSC, CSC-1 and CSC-2 target cells. (E) Inhibition of T RM -cell-mediated killing. CSC-1 cells are preincubated in the presence of isotype control, anti-major histocompatibility complex class I (MHC-I) or anti-intercellular adhesion molecule 1 (ICAM-1) neutralizing monoclonal antibody (mAb) and then CTL were added at 5:1 E:T ratio. Symbols represent replicates and horizontal bars represent means+-SEM (n=3). P value was determined by two-way analysis of variance test. *p

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Effects of TNF on the suppressive function of TNFR2 + Tregs. Purified CD4 + T cells were cultured in medium alone, TNF (50 ng/mL), TNF combined with anti-TNFR2 mAbs (10 ug/mL), or isotype IgG, as indicated, for 72 hours. The representative FACS analysis of (A) CTLA-4 + cells and (B) PD-L1 + cells in TNFR2 + Tregs as indicated, gated on live CD4 + Foxp3 + cells. Summary of the proportions of (C) CTLA-4 + cells (n=3) and (D) PD-L1 + cells (n=3) in Tregs within each condition. Data are expressed as means +- SEM. *, P

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 2 T FR expansion in lymphoid tissues during chronic SIV infection. ( a ) Disaggregated lymph node and spleen cells from SIV uninfected ( n =9) or chronically SIV-infected rhesus macaques ( n =11) were analysed by flow cytometry. Representative examples of flow cytometry gating are shown. Of viable CD3 + CD8 - cells, follicular subsets were defined as CXCR5 + cells (F) and germinal centre subsets were defined as CXCR5 hi PD-1 hi cells (GC). Of these subsets, regulatory cells were defined as CD25 hi CD127 - . T FR (CXCR5 + CD25 hi CD127 - ) were Foxp3 + , whereas T FH (CXCR5 + CD25 lo/- ) were Foxp3 - . ( b ) The percentages of each rhesus macaque regulatory subset, as analysed in a are shown. ( c ) The ratios of each regulatory cell population to its non-regulatory cell counterpart are shown. ( d ) The percentage of total CTLA-4 expression is shown in SIV-uninfected ( n =9) and chronically SIV-infected ( n =8) rhesus macaques. The horizontal bars of each graph indicate the median value and are listed where appropriate for clarity. Statistical analyses were performed by Mann-Whitney (Wilcoxon) tests to compare unpaired, nonparametric values and significance is denoted by asterisks where * P

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 6 Flow cytometry analysis of T cell-/lymphocyte-specific markers on normal and malignant B cells from CLL patients. a Summary scheme representing functional implications of CLL-specific candidate genes selected for flow cytometric analysis. b Flow cytometric analysis of expression of CTLA-4, TIGIT, CD276, LILRB4, and CD2 on peripheral blood B cells of CLL patients. The expression was determined for non-malignant B cells (""Normal""; CD19 + CD5 - B cells, represented in green) and neoplastic B cells (""CLL"", CD19 + CD5 + B cells, represented in orange) detected in the same samples. ""Co,"" no antibody staining control; ""Ab,"" staining with the antibody of interest as indicated. c Normalized median fluorescence intensities (target MFI - MFI of negative control [Co]; nMFI). d Delta normalized median fluorescence intensities between CLL cells and normal B cells (DeltanMFI (CLL-normal)) for each patient tested

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 3 The intracellular AND logic with different signaling domains. a Diagram of intracellular AND logic. b Primary human CD8+ T cells were transduced with FOS zipCAR-containing CD3zeta domain and RR zipCAR-containing CD28 domain. Cytotoxicity against Her2- and Axl-expressing Nalm6 was measured 24 h after adding alpha-Her2-SYN9 and/or alpha-Axl-EE zipFvs. The heatmap indicates cytotoxicity at varying zipFv concentrations ( n = 3, data are represented as mean). c Cytotoxicity of CD8+ T cells transduced with FOS zipCAR-containing CD3zeta domain and RR zipCAR-containing 4-1BB domain. The heatmap indicates cytotoxicity at varying zipFv concentrations ( n = 3, data are represented as mean). d (Left) Isolated Treg cells were transduced with two zipCAR constructs: SYN6-CD3zeta-P2A-FOXP3 and SYN1-CD28-P2A-puro. After puromycin selection (2 mug/mL), Treg cells were co-cultured with Her2- and Axl-expressing Nalm6 target cells (Right) The heatmap shows surface CTLA-4 expression detected after 48 h by flow cytometry at varying zipFv concentrations (alpha-Axl-SYN5 and alpha-Her2-SYN2) ( n = 3, data are represented as mean).